Method of conditioning vascular systems by administering 3,5 dichloroaspirin and method of evaluating pharmaceutical compounds

ABSTRACT

3,5 DICHLOROASPIRIN IS ADMINISTERED ORALLY OR INTRAVENOUSLY TO RENDER MORE NEGATIVE THE SURFACE CHARGE OF THE INTERIOR SURFACE OF THE VASCULAR SYSTEM AND THEREBY TO DECREASE THROMBOTIC TENDENCY THEREIN. This also increases the streaming potential of the vascular interface and increases Lee White clotting times, thrombin times, and partial thromboplastin times and recalcification times. The overall effect is to decrease thrombotic tendencies and to increase blood flow in the extremities. A method is provided for evaluating 3,5 dichloroaspirin and other such compounds.

United States Patent [1 1 Sawyer METHOD OF CONDITIONING VASCULAR SYSTEMSBY ADMINIS'IERING 3,5 DICHLOROASPIRIN AND METHOD OF EVALUATINGPHARMACEUTICAL COMPOUNDS [76] Inventor: Philip Nicholas Sawyer, 606Third St., Brooklyn, NY. 11215 221 Filed: Aug. f6, 1914 211 Appl. No;497,851

[52] US. Cl. 424/230 [5|] Int. Cl. A61K 31/60 [58] Field of Search424/230 [56] References Cited OTHER PUBLICATIONS Chem Abst. 64 126330(I966).

Chem. Abst. 73 646040 1970).

[ Dec. 23, 1975 Primary Examiner-Stanley J. Friedman Attorney, Agent, orFirm-Roberts & Cohen [57] ABSTRACT 3,5 dichloroaspirin is administeredorally or intravenously to render more negative the surface charge ofthe interior surface of the vascular system and thereby to decreasethrombotic tendency therein. This also increases the streaming potentialof the vascular interface and increases Lee White clotting times,thrombin times, and partial thromboplastin times and recalcificationtimes. The Overall effect is to decrease thrombotic tendencies and toincrease blood flow in the extremities. A method is provided forevaluating 3,5 dichloroaspirin and other such compounds.

4 Claims, 6 Drawing Figures METHOD OF CONDITIONING VASCULAR SYSTEMS BYADMINISTERING 3,5 DICHLOROASPIRIN AND METHOD OF EVALUATINGPHARMACEUTICAL COMPOUNDS FIELD OF INVENTION This invention relates topharmaceutical compounds for treating the vascular system and to methodsof evaluating pharmaceutical compounds.

BACKG ROUN D Various cell systems have been investigated in an attemptto explain specificities in cell recognition, rejection and adhesion(Danielli, J. F. Cell to cell interaction. J. Physio. Vol. 98: 109,1940). Evidence indicates that platelets and erythrocytes have commonsurface properties that may greatly influence cell-cell interactions(Seaman, G. V. et al. Cell interaction a surface charge phenomena.Biochem. Biophys, Vol 1 l7, l966; Mehrishi, J. N. Phosphate groups onthe surface of human blood platelets, Nature, Vol. 226, 5244, i970;Davies, D. F., et al. The electrophoretic mobility of RBC a measure ofthe surface activity. Clin. Sci., Vol. l7, 1965). One such property isthe negative surface charge of the membrane, which was first describedby Abramson in 1928 (Abramson, H. A. Electrophoretic migration of inertparticles and blook cells in sols. and gels. Amer. J. of Med. Sci. Vol.167, 1928). Intact cells at a physiological pH tend to move toward ananode as can be shown by quantitative electrophoresis measurements. Theeffect is accounted for by an excess of anionic over cationic groups inthe surface layer that determines electrophoretic mobility (Madoff, M.A. et at. Sialic acid of human blood platelets .I. of Clin. lnv. Vol.43, No. 5, 1964). Platelets have a true iso-electric point at a pH of3.5 to 4.5 which indicates that the net negative charge excess isconsiderable. Upon treatment with neurominidase, 60 percent of thecharge can be removed. apparently due to the removal of sialic acidresidues located on the cell surface. Platelets so treated tend toaggregate spontaneously. This then would appear to indicate thatelectrostatic repulsion is partly responsible for vascular homeostasis(Weiss, L. Biophysical aspects of initial cell interaction with solidsurfaces. Fed. Proc. Vol. 30, No. 5, Sept-Oct, l97l', Salzman, E. Roleof platelets in blood surface interaction Fed. Proc., Vol. 30: No. 5,Sept. Oct., l97l It is possible to cause platelets as well as othercells to aggregate by the addition of positively charged macromoleculessuch as polylysine and protamine sulfate (Hampton, J. R., and Mitchell,J. R. A. Modification of the electrokinetic response of blood platelets.Nature, l966; Massini, P. and Luscher, E. On the mechanism of which cellcontact induces the release reaction of blood platelets, Thromb. etDiath., Vol. 27, I972). It has also been shown that positively chargedmetallic surfaces that exhibit positive potentials vs. NHE inblood/saline are thrombogenic (Sawyer, et al. Relation betweenThrombosis on Metal Electrodes and the Position of Metal in theElectromotive Series, Nature, Vol. 2l5, No. 5109, p. 1494, Sept. 30,I967). Modifications of collagen prostheses to reduce surface negativitypromotes thrombus formation in vivo (Sawyer, et at., Critical potentialfor Thrombus Deposition on Metal Surfaces in Vivo. Vascular surgicalservices and the Electrochemistry and Biophysical Labs. of the Dept. of

2 Surgery and Surgical Research, State Univ. of NY. Downstate MedicalCenter, Bklyn.).

The above supports the view that electrostatic repulsion per se is inpart responsible for platelet noninteraction, at both cell-cell andblood vessel wall-blood cell levels under normal conditions. Oneexplanation of platelet transformation into highly adhesive cellsrelates to surface charge. Metallic tube implants have demonstrated therelevance of surface potential and 0 thrombogenecity (Sawyer et al.,Critical potential for Thrombus Deposition on Metal Surfaces in Vivosupra). Metallic tubes with varying spontaneous potentials in contactwith blood were implanted in dogs. Those tubes which possess ahomogeneous negative surface potential vs NHE in contact with bloodremain patent far longer than did those implants with positivepotentials (Sayer et al., Critical potential for Thrombus Deposition onMetal Surfaces in Vivo supra). Likewise, polymer materials with anexcess of oriented negative charge residues, (i.e., SO, and COO*) incontact with blood also support the premise that a homogeneous negativesurface potential is one criteria for antithrombogenicity (Sawyer etal., Critical potential for Thrombus Deposition on Metal Surfaces inVivo).

Potential dependent cell adhesion characteristics were first observed inI963. The fact has been established, both in vitro and in vivo, thatanodic potentials imposed upon a P1 wire produced cell precipitation.The effects of varying pH and heparin were also established. Resultshave demonstrated that an acidic pH potentiates cell adhesion, while abasic pH inhibits cell adhesion. A series of experiments produced a semiquantitative method of evaluating cell adhesion in a highly ordered andcontrolled environment.

Data is available which correlates potential dependent cell adhesionwith the influence of various pharmacological agents on plateletadhesion and thrombus formation on vascular prostheses. Furthermore,various pharmacological agents have been shown to modify thrombusdeposition in vivo on metallic prostheses, the injured blood vessel walland collagen implants (Sawyer et at, Utility of Anticoagulant Drugs inVascular Thrombosis: Electron Microscopic and Biophysical Study,Surgery, St. Louis, Vol. 74, No. 2, PP. 263-275, August, i973).

SUMMARY OF INVENTION It is an object of the invention to provide animproved technique for the treatment of vascular systems of warm bloodedanimals.

lt is another object of the invention to provide a new technique forrendering more negative the intimal surface charge of the vascularsystem.

Yet another object of the invention is to provide an improved techniquefor decreasing thrombotic tendency in vascular systems.

Still another object of the invention is to provide an improvedtechnique for decreasing the thrombotic tendency in vascular systems asmanifested by an increase in the streaming potential of the vascularinterface, an increased clotting time, thrombin time, partialthromboplastin time, and recalcification time.

To achieve the above and other objects of the invention, there isprovided a method which comprises administering 3,5 dichloroaspirin to awarm blooded animal to render more negative the surface charge on theinterior surface of the vascular system and thereby decrease thrombotictendencies therein. The 3,5 di- 3 chloroaspirin has the followingstructure COOH C OCH 3 c1 CI The 3,5 dichloroaspirin may in accordancewith the invention be administered orally. It is preferably administeredin an amount in the order of magnitude of about 15 mg./kg. of bodyweight daily.

The 3,5 dichloroaspirin may also be administered intravenously inaccordance with the invention in an amount in the order of magnitude ofabout mg./kg. of body weight daily.

Other objects, features and advantages of the invention will be found inthe detailed description which follows hereinafter.

BRIEF DESCRIPTION OF DRAWING In the drawing:

FIG. 1 diagrammatically illustrates apparatus for conducting tests inaccordance with the invention:

FIGS. 2(a) and 2(1)) are charts indicating potential dependent plateletadhesion vs. surface potential in the apparatus of FIG. 1;

FIG. 3 illustrates ADP aggregation levels vs. surface potentialaggregation levels in chart form; and

FIGS. 4(a) and (b) illustrate platelet dependent cell adhesion vs.surface potential.

DETAILED DESCIPTION As shown in FIG. I, a Bausch & Lomb movable stagemicroscope with phase contrast optics and an oil immersion lens wasemployed. The optics include a phase microscope using an oil immersionlens at 1000 magnification. The microscope is focused on one small areaof the platinum electrode in order to observe platlet sticking. A lucitecell or chamber 12 with a volume of 2 ml was constructed so as to allowthe easy insertion of a Pr wire electrode 14 and two salt bridges l6 andI8 (agar bridges used as electrolytic conductors).

A Pt electrode with surface properties that are well defined was chosenas the test surface. Of great importance in obtaining accurate resultsin cleanliness (demonstrably free of blood coagulation factors and otherproteins or chemicals which might excite blood clotting), particularlyof the lucite cell and the electrodes. To achieve the above, the cell isfirst rinsed with distilled water and washed with a mild soap solution.The cell is then treated with a solution of 50 percent water and 50percent methanol in order to denature any proteins or accumulatedthrombin. It is finally rinsed with triple distilled water and allowedto dry.

The electrode is cleaned first by immersion in nitric acid for at least15 minutes and then flamed. It is then inserted into the lucite cell.The cell is filled with a protein-free plasma-like solution, namelyKrebs l solution (Sawyer et al.. Electrochemical Precipitation of HumanBlood Cells and its Possible Relation to Intravascular Thrombosis,Proceedings of the National Academy of Sciences. Vol. 51. No. 3. PP.428-432, March. 1964). The electrode is further cleaned by passing acathodic current and degassing the electrodev The modified Krebs 1solution is then removed and fresh solution is added for actualexperimentation.

Ten ml of human blood are drawn into a sterile syringe and placed intotwo citrated vacutainers. The blood is centrifuged at 1000 rpm for 20minutes. This produces a supernatant plasma with a high plateletconcentration. 0.5ml of platelet rich plasma (PR?) is then added to thecell already containing 1 ml of the Krebs 1 solution which is thenfilled (approximately two milliliters) for the purpose of obtaining aseries of control values.

In determining the effects of pharmacological agents on the adhesivebehavior of platelets, varying amounts of the test drug are added to theabove solution. The experiment is run at a pH of 7.35. Tenth normal HCIand NaOH buffer solutions were used for buffering to alter the pH range.

The salt bridges connect with ionic solutions in vessels 20 and 22. Theionic solutions may be, for example, saturated potassium chloridesolution or a solution of sodium chloride. A potentiostat 24 was used tocontrol the surface potential of the Pt wire. Current was read by meter26 and voltage was read by meter 28. First the potential was set at 6()Omv vs. NHE and allowed to remain there for 2 minutes. A count ofplatelets sticking to the electrode was then taken. The potential wasthen increased in steps of mv and another count taken 2 minutes later.This procedure was repeated until the potential was brought through 0 to+600 mv vs. NHE (normal hydrogen electrode). The initial potential wasnot set below 600 mv vs. NHE in an effort to avoid the formation of gasbubbles on the electrode.

After each run, the apparatus is cleaned as previously described andanother run is made. Upon completion of a series of experiments, thenumber of adherent platelets are averaged at each potential and adeviation from the mean is tabulated. The experimental results can bestbe represented by graphs, where the number of platelets adhering to theelectrode is plotted against potential of the electrode under varyingexperimental conditions.

Included among the agents evaluated were (a) three anticoagulants,namelyz. heparin, aspirin (ASA), and 3,5 dichloroaspirin, (b)coagulants, namely protamine sulfate and chlorpromazine, and (c) aproteolytic en'- zyme (brinase); code number herein C The drugconcentrations used (except for 3.5 dichloroaspirin which was notpreviously used) were in most cases ten times less than normal, normaland ten times greater than normal with respect to a clinical therapeuticdosage. The term normal as used herein means the average concentrationnormally found in plasma when the drug is administered orally andimplies the maximum normal blood level with non toxic effect by whateverroute the material is administered.

On the basis of ten independent blood samples, a standard curve (seeFIGS. 2(a) and 2(b)) was arrived at for the potential dependent adhesioncharacteristics of human platelets on P! wire. Analysis of the controlgraph shows a peak in the curve at 40() mv. NHE, far below the plateauregion which starts at the anodic potential of +400 mv NHE. It is thispoint in the curve where one observes reversible migration of plateletstoward the electode followed by subsequent primary stage plateletadhesion. This reversible aggregation phenomena also occurs in vivo withthe blood vessel wall and has been reported in other test systems.evaluating platelet adhesion (Fulher, M. B., et al. Reversiblealterations in platelet morphology produced by anticoagulants and coldblood. vol. 9, 602, 1954). One cannot, however, actually say whether thecharge on the metal at this potential is comparable to the chargedensity of an equal area of the blood vessel wall.

At a potential near the point of zero charge (PZC of the Pt electrodewhat appeared to be first stage viscous metamorphosis (agglutination)was observed. This same observation was made in previously reportedresearch, utilizing a mercury drop electode. In an attempt to establishwhether the point of zero charge and any anodic potential thereafter wasinducing the platelet release reaction, a series of experiments wasconducted using ADP (adenosine diphosphate) results in aggregationcurves utilizing known optical techniques. Stated otherwise, in order todetermine if platelets or blood cell precipitation was related to pointof zero charge, a series of experiments was carried out measuring theprecipitation potential of blood cells on electrodes constructed ofdifferent metals. The available evidence indicates that the depositionof cells on the electrodes always occurred at approximately the samepotential with respect to the normal hydrogen electrode rather thanbeing related to the point of zero charge of the materials in question.

A series of experiments was carried out matching the effects of ADP andthe anodic electrode on pertinent aggregation comparing increasinglyanodic potentials to the two increasing concentrations of ADP. Thusplatelet aggregation decreased turbidity at various concentrations ofADP in the cell was compared with anodic potential to produce the samedegree of aggregation, that is, decreased turbidity.

Following this, samples of PRP (platelet rich plasma) which had beenexposed to various anodic potentials for a period of 3 minutes weremasured for optical density changes compared with no current control(FIG. 3). By inference, comparing the slope of the control curve,produced by varying concentrations of ADP with the curve produced byanodic potentials, the possible correlation between the anodicpotentials, inducing platelet release reaction, through the release ofendogeneous ADP, and ADP addition to the cell can be compared.

As one approaches the anodic region of the control curve there is arapid increase in the number of cells adhering to the electrode. Visualobservations indicate that second stage platelet adhesion is occurringat appoximately +400 mv NHE. Beyond this, the rate of increase in thenumber of cells adhering to the electrode levels off.

Experiments were conducted comparing heparin, a potent anticoagulant, asa standard and brinase(C a potent proteolytic agent (very capable oflysing proteins). The effect observed with these drugs is plotted inFIG. 2(a). It demonstrates a large reduction in the number of cellsadhering to the electrode regardless of the surface potential. Theresults seem to suggest that heparin is reducing the adhesive behaviorof the plate' lets through an absorption phenomenon. The chemicalstructure of heparin, which is a repeating polymer of mucopolysaccharideunits with an abundance of SO, groups represents an ideal compound forincreasing the mutual electrostatic repulsive forces between the cellsand foreign surface, if absorption takes place. The

results with ASA and with 3,5 dichloroaspirin demonstrate a similartrend. The number of cells adhering to the surface of the electrode fallfar below the number adhering in the control situation in the anodicregion. At no time was any viscous metamorphosis (agglutination)observed with the addition of ASA. This might be explained by the dataof other reseachers who suggest that ASA acetylates the plateletmembrane and by blocking ADP release, suppresses the secondary wave ofplatelet aggregation in in vitro studies (Stemerman, M. B. Drugs,platelets and vascular injury, Proceedings of the 4th Inter. HemostasisConference No. 23, I972; Mustard, 1. F., et al. Modification of plateletfunction, Ann. of the N.Y. Acad. of Sci., Vol. ZOI, I972). The resultswith C the proteolytic enzyme, are conventional. Some cell lysis wasobserved. Almost no cell adhesion was seen with varying surfacepotentials.

The results with protamine sulfate (neutralizing agent for heparin)demonstrate (FIGS. 4(a) and 4(b) a marked increase in the rate andnumber of cells adhering to the electrode. What is observed, uponanalysis of the curve, is that there is a shift in the potential atwhich adhesion occurs, i.e., from +400 mv NHE to 200 mv NHE, with analmost linear relationship be tween potential versus number of cellsadhering to the Pt surface. The skeletal proteins of the plateletmembrane provide a number of possible sites for bond formation withmacromolecular (e.g., COO, NH and SH) groups. The absorption ofprotamine sulfate onto the platelet membrane through disulfide bridgingor due to an acid-base level reaction between the COOH and amine groupscan explain the observed behavior.

Chlorpromazine has been reported to block the glucosyltransferasereaction reported to be the basis for platelet adhesion to an organicsubstrate. The results obtained by the present experiments indicate aconflict. Chlorpromazine seems to enhance the adhesion characteristicsof platelets. Here again one is not sure of the mechanism.Chlorpromazine may be involved metabolically, producing an externalalteration thus enhancing platelet adhesion in the absence of collagen.

The data presented herein tends to support the promise that mutualelectrostatic repulsion between blood platelets and the arterial wall isjust one of the forces responsible for homestatis in the blood vascularsystem (Born, GVR. Current ideas on the mechanism of plateletaggregation, Ann of the NY. Acad. of Sci., Vol. 201, I972). The resultsindicate that anodic surface potentials enhance platelet adhesion toappropriately charged foreign surfaces in vitro. Visual observationslink first stage viscous metamorphosis with a point near the point ofzero charge of the metal. Maximum platelet adhesion occurs on a PI wireat a surface potential of +400 mv NHE.

Various anticoagulants such as ASA, 3,5 dichloroaspirin and heparinappear to alter the adhesion charac teristics of platelets by a simpleadsorption process. Both protamine sulfate and chlorpromazine enhancethe adhesive characteristics of platelets. In the latter case theresults cannot be explained by a simple adsorption phenomenon, andmetabolic involvement must be indicated. This test system seems to be auseful one in evaluating the effects of various pharmacological agentsupon platelet adhesion characteristics in association with foreignsurfaces.

The structure of 3,5 Dichloroaspirin is COOl-I C OCH3 Results using 3,5dichloroaspirin are shown in the following table (reference being madeto Applicants *3.5 Dichloroaspirin Streaming Potential (in vivo)Electrtmsmosis Electrophoresis in vivo dogs Zeta Potential CanineVessels In Vivo Canine Platelets RBCs jugular artery rt artery lt IntimeAdventitia Zeta Potential vein femoral Artery IS!) IT] (7) ControlControl 24 hrs. l8.7 control U.l J.5 ().25 Control Vein 7.l 5.l (3) 76hrs l6.9 crush +0.2 +0.5 Fogarty +0.5

3.5 l 3,5 Dichloro- Dichloro control ll.25 l .0 AH) 3.5 aspirin aspirincrush 4).l U.5 Dichloro- Artery HM I315 (2) 300 mg/ 31M) mg/ Fogarty().5 aspirin day da 3(lllmg/ 2 day 24 hrs l7.l control tl.5 (l.h ().Sorally Vein 9.0 13.5 (I) 76 hrs l8.2 crush l.2 (l.h Fogarty t).5

Coagulation Study Rat Mesentry TR APTT RCT Occlusion Current 65 MicroAmp sion 65 Micro Amp Femoral Femoral Total occlusion Time vein ArteryControl 48.0 r min 3 Control control U.h (l.l lst day 5.9 l .0 20.5crush ().25 ().3 4th day 6.6 ...(i 83.1 3,5 Fogarty +(l.l (l. lDichloroaspirin 3.5 5mg/kg *Signs refer to the potential atDichloroaspirin drug given the interface. 300 mg) day oral l.V. A: 93.0:31.1) min.

lst day 5.3 lfi.5 74.4 hr. before 4th day 5.9 2L4 78.4 lst day 7.3 17.498.7 4th day 6.! 33.3 l()l.() Smg/kg lst day 7 h l2.2 3 l .5 IV. daily70.0: 13 mins. 4th day 5 days prior US. Pat. No. 3,722,504).

Regarding the above electrochemical screen measurements of the caninevessel. tests on streaming potential under control conditions and incomparison with aspirin, electroosmosis of canine artery and veins whileusing this material. platelet electrophoresis determinations. the effectof 3.5 dichloroaspirin on the charge characteristics of platelets andred cells as indicated. coagulation studies and measurements of the timebut increases the partial thromboplastin time (PTI) and therecalcification time (RCT) of blood from the four dogs in which thestudies were carried out. Five milligrams per kilogram intraperitonealyonehalf hour before the study and 5 mgs/Kg intraperitonealy for each of5 days before the test prolonged rat mesentery occlusion time from acontrol level of 48 minutes for a single intraperitonealy dose andminutes for a five day intraperitoneal dose.

Data indicates that 3.5 dichloroaspirin has a dramatic effect on theblood-vascular interface. Rather limited effects which are not entirelyexplained are seen. by electroosmosis of canine blood vessels, inanimals given 300 mgs. per day orally and rather marked effects onplatelets. The dramatically increased coagulation times and RC'Tindicated that it has very fundamental effects on the coagulationenzymes.

A summary of its physiologic effect as shown by rat mesentery studiesdemonstrate prolonged rat mesentery electrical thrombosis times andtherefore it has profound antithrombotic effects on the intactmicrocirculation.

2. A method as claimed in claim 1 comprising administering the 3,5dichloroaspirin orally or intraperitonealy to increase the streamingpotential of the vascular interface and to increase clotting time,thrombin time, partial thromboplastin time and recalcification time.

3. A method as claimed in claim 2 wherein the 3,5 dichloroaspirin isadministered orally in an amount in the order of magnitude of about 15mg./kg. of body weight daily.

4. A method as claimed in claim 2 wherein the 3,5 dichloroaspirin isadministered intraperitonealy in an amount in the order of magnitude ofabout 5 mg./kg. of body weight daily.

1. A METHOD FOR RENDERING MORE NEGATIVE THE INTERNAL SURFACE OF AVASCULAR SYSTEM IN ORDER TO DECRESE THROMBOTIC TENDENCY, SAID METHODCOMPRISING ADMINISTERING 3,5 DICHLOROASPIRIN IN THE ORDER OF MAGNITUDEOF ABOUT 5-15 MG./KG OF BODY WEIGHT DAILY TO A HOST REQUIRING SUCHTREATMENT, THE 3,5 DICHLOROASPIRIN HAVING THE STRUCTURE
 2. A method asclaimed in claim 1 comprising adMinistering the 3,5 dichloroaspirinorally or intraperitonealy to increase the streaming potential of thevascular interface and to increase clotting time, thrombin time, partialthromboplastin time and recalcification time.
 3. A method as claimed inclaim 2 wherein the 3,5 dichloroaspirin is administered orally in anamount in the order of magnitude of about 15 mg./kg. of body weightdaily.
 4. A method as claimed in claim 2 wherein the 3,5 dichloroaspirinis administered intraperitonealy in an amount in the order of magnitudeof about 5 mg./kg. of body weight daily.